Isolated populations of renal stem cells and methods of isolating and using same

ABSTRACT

Isolated populations of fetal renal stem cells and progenitor cells are provided. Also provided are methods of generating and using these isolated populations of cells.

FIELD AND BACKGROUND OF THE INVENTION

The present invention, in some embodiments thereof, relates to isolatedpopulations of renal stem cells and methods of isolating and using same.

The kidney is a vital organ in mammals, responsible for fluidhomeostasis, waste excretion, and hormone production. There are avariety of possible injuries and disorders including cancer, trauma,infection, inflammation and iatrogenic injuries or conditions that canlead to chronic disease or cause reduction or loss of function of akidney. The incidence of chronic kidney disease in the United States hasreached epidemic proportions, and a significant number of these patientswill develop end-stage renal disease (ESRD), with glomerular filtrationrates too low to sustain life. Dialysis is the major treatment modalityfor ESRD, but it has significant limitations in terms of morbidity,mortality, and cost. Allogenic kidney transplantation providessignificant benefits in terms of mortality and is ultimately lesscostly, but is hampered by a severe shortage of available donor organs.Acute renal failure (ARF) is also quite common, having a mortality ratethat ranges from 20 to 70%. For a number of reasons, includingaggressive care of an older patient population, the mortality rate dueto ARF has not changed over the past 20 years despite advances intechnology and therapies.

Although kidney disease has a variety of individual types, they appearto converge into a few pathways of disease progression. The functionalunit of the kidney is the nephron. There is a decrease in functioningnephrons with the progression of the disease; the remaining nephronscome under more stress to compensate for the functional loss, therebyincreasing the probability of more nephron loss and thus creating avicious cycle. Furthermore, unlike tissues such as bone or glandularepithelia which retain significant capacity for regeneration, it hasgenerally been believed that new nephron units are not produced afterbirth, that the ability of the highly differentiated tissues andstructures of the kidneys have limited reparative powers and, therefore,that mammals possess a number of nephron units that can only declineduring post-natal life. There is an increasing interest in developingnovel therapies for kidney disease, including artificial organs, geneticengineering, and cell therapy.

The early development of the mammalian metanephros, the direct precursortissue of the adult kidney, is a complex process that involves highlyregulated interactions between two derivatives of the intermediatemesoderm, the wolffian duct and the metanephric/nephrogenic mesenchyme.Reciprocal signaling between the neohrogenic/metanephric mesenchyme anda derivative of the nephric duct known as the ureteric bud results inbranching of the ureteric bud (UB) and condensation of metanephricmesenchyme (MM) at its tips (4, 5). The condensed mesenchyme is thoughtto form a precursor cell population, which both maintains itself at thetips of the UB (via proliferation and/or addition from the surroundingnon-condensed mesenchyme) and gives off cells that differentiate intonephrons, the functional filtration unit of the kidney (6). Recentexperiments have established that the progenitor cell in the MM fulfilsthe criteria of a true committed stem cell in that is capable ofself-renewing and of differentiating towards different types of nephronepithelia (7-9).

The human metanephros appears at the 5^(th) week of gestation and renalstem/progenitor cells in the nephrogenic mesenchyme are induced to formnephrons until 34 weeks of gestation (4, 6). For renal regeneration,both human precursor tissue (10-12) or fetal kidney cell transplantation(13, 14) can be utilized. Isolation of specific human renal progenitorsfrom the nephrogenic mesenchyme requires the characterization of surfacemarkers that would enable cell collection. Given the cellularheterogeneity in the developing human kidney (6), eliminating theunwanted mature cell populations from further cultivation steps, priorto transplantation, would increase the purity of the graft and allow fora better defined cell composition to be transferred.

While the transcriptional program specifying a renal progenitor cell hasbeen thoroughly contemplated (15) corresponding cell surface markershave been hardly studied. Recently, the present inventors performedmicroarray studies of the human kidney, including adult (AK) and fetalkidneys (FK) and their corresponding tumors, renal cell carcinoma (RCC)and wilms' tumor (WT) (16). Wilms' tumor is classified as a primitive,multilineage malignancy of embryonic renal precursors that are arrestedin different stages of differentiation, thus forming in the tumor a cellpopulation similar to condensed mesenchyme (blastema) and also matureepithelial/tubular and stromal cells (17). While fetal kidneys wereheterogeneous, WT xenografts were used that by serial passage in micewere highly enriched for blastema at the expense of differentiatedelements (16, 18). Genes that were up-regulated in both the stem-like WTxenografts and the human FK were sought, as these were suggested tocharacterize the progenitor population arising from the MM (‘progenitor’genes). Among these were the transcription factors specifying the kidneyprogenitor cells (7, 15, 19, 20) including WT1, PAX2, LIM1, SIX1, EYA1,SALL1, and CITED1. In addition, various cell surface markers weredetected, including NCAM1, ACVRIIB, FZD2, FZD7, GPR39, NTRK2 andDLK1/PREF (16).

U.S. Patent Application 20020102241 discloses Flk-1 positive/Sca-1negative adult renal stems cells and uses thereof. The cells aredescribed as useful for the regeneration of damaged kidney tissue, thegeneration of artificial kidneys and the delivery of transgenes.

U.S. Patent Application 20050260623 discloses the identification ofadult human stem cells including adult renal stem cells by detecting theexpression of Oct-4, and the lack of GJIC activity.

U.S. Patent Application 20070065942 provides human renal stem cells.Also described are human renal stem cells isolated from the papillaryregion of the human kidney and methods of isolating the same. Alsodescribed are methods for culturing, characterizing, and differentiatingthe same, including methods for identifying human renal stem cells thatare positive for Nestin and CD133, and methods for allowing the cells todifferentiate into neurons.

Chang, et al., (1987), Cancer Res., 47:1634-1645 teach a method of fetalrenal stem cell isolation, based on the cell's contact insensitivity.

Gibson-D′ ambrosio et al [In Vitro Cell Dev Biol. 1987 April;23(4):279-87] teach heterogenic population of cells which may compriserenal stem cells. It is stated that these cells in culture are proximaltubule epithelial cells, indicating that these are in factdifferentiated cells and not stem cells.

WO/2005/021738 teaches methods for isolation of kidney stem cells, cellsisolated by the methods, and therapeutic uses for those cells. Morespecifically, the invention relates to isolated kidney-derivedprogenitor cells that have the potential to differentiate to form cellsof any one or all three germ cell layers (endoderm, mesoderm, ectoderm),as well as methods for isolating the cells and for inducing specificdifferentiation of the cells isolated by the method, and specificmarkers that are present in these cells such as proteins andtranscription factors. Also described are NCAM negative cells.

SUMMARY OF THE INVENTION

According to an aspect of some embodiments of the present inventionthere is provided a method of isolating renal stem cells, the methodcomprising enriching for a subpopulation of renal cells from a fetalrenal tissue, the subpopulation of renal cells having a NCAM+ signature,wherein the enriching is effected such that at least 80% cells are ofthe subpopulation of renal cells.

According to some embodiments of the invention, the enriching iseffected by detecting surface marker expression of NCAM.

According to some embodiments of the invention, the detecting furthercomprises detecting surface marker expression of EpCAM.

According to some embodiments of the invention, the method furthercomprises isolating cells having an EpCAM−/NCAM+ signature.

According to some embodiments of the invention, the method furthercomprises isolating cells having an EpCAM+/NCAM+ signature.

According to some embodiments of the invention, the method furthercomprises culturing the subpopulation of renal cells in serum freemedium following the enriching.

According to an aspect of some embodiments of the present inventionthere is provided a method of isolating MM stem cells the methodcomprising enriching for a subpopulation of renal cells from a fetalrenal tissue, the subpopulation expressing a EpCAM−/FZD7+ signature,wherein the enriching is effected such that at least 80% cells are ofthe subpopulation of renal cells.

According to some embodiments of the invention, the enriching iseffected by detecting surface marker expression of EpCAM and FZD7.

According to some embodiments of the invention, the detecting furthercomprises detecting surface marker expression of NCAM.

According to some embodiments of the invention, the method furthercomprises isolating cells having an EpCAM−/FZD7+/NCAM− signature.

According to some embodiments of the invention, the method furthercomprises isolating cells having an EpCAM−/FZD7+/NCAM+ signature.

According to some embodiments of the invention, the method furthercomprises culturing the subpopulation of renal cells in serum freemedium following the enriching.

According to an aspect of some embodiments of the present inventionthere is provided a method of isolating renal stem cells the methodcomprising enriching for a subpopulation of renal cells from a fetalrenal tissue, the subpopulation of renal cells having a NCAM+/EpCAM+signature, wherein the enriching is effected such that at least 80%cells are of the subpopulation of renal cells.

According to some embodiments of the invention, the enriching iseffected by detecting surface marker expression of NCAM and EpCAM.

According to some embodiments of the invention, the detecting furthercomprises detecting surface marker expression of a marker selected fromthe group consisting of FZD7, NTRK and PSA−NCAM.

According to some embodiments of the invention, the method furthercomprises culturing the subpopulation of renal cells in serum freemedium following the enriching.

According to an aspect of some embodiments of the present inventionthere is provided a method of isolating renal stem cells the methodcomprising enriching for a subpopulation of renal cells from a fetalrenal tissue, the subpopulation of renal cells having a NCAM+/FZD7−signature, wherein the enriching is effected such that at least 80%cells are of the subpopulation of renal cells.

According to some embodiments of the invention, the enriching iseffected by detecting surface marker expression of NCAM and FZD7.

According to some embodiments of the invention, the detecting furthercomprises detecting surface marker expression of EpCAM.

According to some embodiments of the invention, the method furthercomprises culturing the subpopulation of renal cells in serum freemedium following the enriching.

According to an aspect of some embodiments of the present inventionthere is provided a method of isolating renal stem cells, the methodcomprising enriching for a subpopulation of renal cells from a fetalrenal tissue, the subpopulation of renal cells having a EpCAM+/FZD7+signature, wherein the enriching is effected such that at least 80%cells are of the subpopulation of renal cells.

According to some embodiments of the invention, the enriching iseffected by detecting surface marker expression of EpCAM and FZD7.

According to some embodiments of the invention, the detecting furthercomprises detecting surface marker expression of NCAM.

According to some embodiments of the invention, the method furthercomprises culturing the subpopulation of renal cells in serum freemedium following the enriching.

According to an aspect of some embodiments of the present inventionthere is provided a method of isolating renal cells, the methodcomprising enriching for a subpopulation of renal cells from a fetalrenal tissue, the subpopulation of renal cells having aNCAM−/EpCAM+/FZD7− signature, wherein the enriching is effected suchthat at least 80% cells are of the subpopulation of renal cells.

According to some embodiments of the invention, the enriching iseffected by detecting surface marker expression of EpCAM, NCAM and FZD7.

According to an aspect of some embodiments of the present inventionthere is provided a method of isolating renal cells, the methodcomprising enriching for a subpopulation of renal cells from a fetalrenal tissue, the subpopulation of renal cells having aNCAM−/EpCAM+/CD24+/CD133+ signature, wherein the enriching is effectedsuch that at least 80% cells are of the subpopulation of renal cells.

According to some embodiments of the invention, the enriching iseffected by detecting surface marker expression of EpCAM, NCAM CD24 andCD133.

According to an aspect of some embodiments of the present inventionthere is provided an isolated population of cells comprising at least80% fetal renal stem cells having a EpCAM−/FZD7+ signature.

According to some embodiments of the invention, the renal stem cellshave a EpCAM−/FZD7+/NCAM− signature.

According to some embodiments of the invention, the renal stem cellshave a EpCAM−/FZD7+/NCAM+ signature.

According to an aspect of some embodiments of the present inventionthere is provided an isolated population of cells comprising at least80% fetal renal stem cells having a NCAM+ signature.

According to an aspect of some embodiments of the present inventionthere is provided an isolated population of cells comprising at least80% fetal renal stem cells having a NCAM+ EpCAM− signature.

According to some embodiments of the invention, the fetal renal cellsfurther comprise an EpCAM− signature.

According to an aspect of some embodiments of the present inventionthere is provided an isolated population of cells comprising at least80% fetal renal stem cells having a ALDH+ signature.

According to an aspect of some embodiments of the present inventionthere is provided an isolated population of cells comprising at least80% MM-derived fetal progenitor cells having a NCAM+/EpCAM+ signature.

According to some embodiments of the invention, the MM-derivedprogenitor cells further express a surface marker selected from thegroup consisting of FZD7, NTRK and PSA−NCAM.

According to an aspect of some embodiments of the present inventionthere is provided an isolated population of cells comprising at least80% fetal renal stromal cells having a NCAM+/FZD7− signature.

According to some embodiments of the invention, the renal stromal cellshave a EpCAM−/NCAM+/FZD7− signature.

According to an aspect of some embodiments of the present inventionthere is provided an isolated population of cells comprising at least80% fetal ureteric bud cells having a EpCAM+/FZD7+ signature.

According to some embodiments of the invention, the ureteric bud cellshave a EpCAM+/FZD7+/NCAM− signature.

According to an aspect of some embodiments of the present inventionthere is provided an isolated population of cells comprising at least80% fetal renal cells having a NCAM−/EpCAM+/FZD7− signature.

According to an aspect of some embodiments of the present inventionthere is provided an isolated population of cells comprising at least80% fetal renal cells having a NCAM−/EpCAM+/CD24+/CD133+ signature.

According to an aspect of some embodiments of the present inventionthere is provided a cell culture comprising a culture medium and any ofthe isolated population of cells of the present invention.

According to some embodiments of the invention, the cells are seeded ona scaffold.

According to an aspect of some embodiments of the present inventionthere is provided a method of treating a renal damage in a subject inneed thereof comprising administering to the damaged kidney of thesubject a therapeutically effective amount of any of the isolatedpopulation of cells of the present invention, thereby treating the renaldisease in the subject.

According to an aspect of some embodiments of the present inventionthere is provided a method of identifying an agent capable of regulatingdifferentiation of a renal stem cell, the method comprising contactingany of the isolated population of cells of the present invention with anagent, wherein a change in developmental phenotype is indicative of theagent capable of regulating differentiation of the renal stem cells.

According to an aspect of some embodiments of the present inventionthere is provided a method of enriching for renal stem cells, the methodcomprising culturing a population of fetal renal cells in a culturemedium devoid of serum, thereby enriching for renal stem cells.

According to some embodiments of the invention, the culturing iseffected by culturing a single cell of the population of fetal renalcells in a single container.

According to some embodiments of the invention, the method furthercomprises selecting a population of fetal renal cells which has a NCAM+signature prior to the culturing.

According to some embodiments of the invention, the population of fetalrenal cells has a NCAM+/EpCAM-signature.

According to some embodiments of the invention, the population of fetalrenal cells has a NCAM+/EpCAM+ signature.

According to some embodiments of the invention, the method furthercomprises selecting a population of fetal renal cells which has a ALDH+signature prior to the culturing.

Unless otherwise defined, all technical and/or scientific terms usedherein have the same meaning as commonly understood by one of ordinaryskill in the art to which the invention pertains. Although methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of embodiments of the invention, exemplarymethods and/or materials are described below. In case of conflict, thepatent specification, including definitions, will control. In addition,the materials, methods, and examples are illustrative only and are notintended to be necessarily limiting.

BRIEF DESCRIPTION OF THE DRAWINGS

Some embodiments of the invention are herein described, by way ofexample only, with reference to the accompanying drawings. With specificreference now to the drawings in detail, it is stressed that theparticulars shown are by way of example and for purposes of illustrativediscussion of embodiments of the invention. In this regard, thedescription taken with the drawings makes apparent to those skilled inthe art how embodiments of the invention may be practiced.

FIGS. 1A-J are photographs illustrating immunostaining of SIX2, NCAM1,FZD7, ACVR2B and NTRK2 in paraffin embedded sections of HFK (12-19 weeksof human gestation); (1A-B) localization of SIX2 to the MM,predominantly to the CM. (C-D) predominant staining of NCAM1 in the MM(including CM) and its derivatives (S- and comma-shaped bodies) andrenal stroma, but not mature tubules or UBs. (E-F) FZD7 demonstratespreferential localization to the nephrogenic zone including MM and itsderivatives, UBs, and newly forming tubules but not the stroma. (G-H)ACVRIIB immunostaining demonstrates predominant expression in thenephrogenic cortex; MM and its derivatives (S and comma shaped bodies),UBs, parietal epithelium of fetal glomeruli but not in the stroma. (I-J)NTRK2 is detected in the MM (including condensates) and its derivatives,UBs and some differentiated tubules. Figures c, e and g are shown in lowmagnification (original ×4), FIGS. 1A, B, D, F and H-J are shown inhigher magnifications (original ×40; I, original ×20).

FIGS. 2A-I are representative flow-cytometry histograms of surfacemarker molecules (green) EpCAM (FIG. 2A), NCAM1 (FIG. 2B), NTRK2 (FIG.2C), CD34 (FIG. 2D), PSA−NCAM1 (FIG. 2E), FZD7 (FIG. 2F), CD90 (FIG.2G), CD24 (FIG. 2H), CD133 (FIG. 2I), and their respective isotypecontrols (red) in HFK (21 weeks of gestation).

FIG. 2J is a summarizing bar graph of single marker staining in HFK(17-21 weeks of gestation). Data were calculated as average % ofexpressing cell±SD. Each marker was tested in 10 HFK.

FIG. 3A is a representative zebra graph of EpCAM staining and thesubpopulation gating. EpCAM subpopulations were gated according to EpCAMstaining intensity (negative, dim or bright) versus FSC.

FIGS. 3B-3D, 3F-3H, 3J-3L, 3N-3P, 3R-3T and 3V-3X are representative dotplot graphs of NCAM1 (3B-3D), PSA−NCAM (3F-3H), FZD7 (3J-3L), NTRK2,(3N-3P), CD24 (3R-3T) and CD133 (3V-3X) expression levels in EpCAMsubpopulations of HFK. Quadrates were placed according to isotypecontrol confiding the negative staining to the lower left quadrant.Percentage of cells in each subgroup appears on the lower rightquadrant.

FIGS. 3E, 3I, 3M, 3Q, 3U and 3Y are summarizing bar graphs of NCAM1(3E), PSA−NCAM (3I), NTRK2 (3M), FZD7 (3Q), CD24 (3U) and CD133 (3Y)expression levels in EpCAM subpopulations. Data are average % of cellsin each subgroup±SD. Analysis of each marker was performed at leastthree times.

FIGS. 4A-B are representative dot plot graphs of EpCAM staining. Cellswere gated in two groups: EpCAM negative (neg) and EpCAM positive (pos)versus FSC.

FIGS. 4C-4L are representative dot plot graphs of PSA−NCAM (4C-D), FZD7(4G-H), NTRK2 (4K-L), CD24 (4E-F) and CD133 (4I-J) co-staining with NCAMin EpCAM positive or negative populations of mid-gestation HFK.Quadrates were placed according to the isotype control confiding thenegative staining to the lower left quadrant. Percentage of cells foreach quadrant appears in the quadrant.

FIGS. 4M-4O are representative dot plot graphs of CD24 and CD133co-staining in EpCAM subpopulations of HFK. Quadrates were placedaccording to the isotype control confiding the negative staining to thelower left quadrant. Percentage of cells for each marker combinationappears in the quadrant.

FIG. 4P is a summarizing bar graph of CD24 and CD133 co-staining inEpCAM Subpopulations. Data are average % of cells in each subgroup±SD.Analysis of each marker was performed at least three times.

FIGS. 5A-L are photographs illustrating immunostaining of FZD2, GPR39,DLK1, CD34, CD90 and CD24 in paraffin embedded sections of HFK (12 or 19weeks of gestation); (A-B) FZD2 immunostaining demonstrates widespreadstaining of renal tubules. (C-D) GPR39 immunostaining demonstratesubiquitous expression in differentiated renal tubular and to a lesserextent in componenets of the nephrogenic cortex. (E-F) Dlk1immunostaining demonstrates ubiquitous expression in differentiatedrenal tubular but not in MM and its derivatives renal, UBs or stroma.(G-H) CD34 immunostaining demonstrates exclusive localization toendothelial cells (glomerular and peri-tubular) in all parts of the HFK,including in the nephrogenic cortex. (I-J) CD90 immunostainingdemonstrates predominant staining in renal tubular cells but not in MMand its derivatives, UBs or stroma. (K-L) CD24 immunostainingdemonstrates widespread expression in mature tubules. Figure (C) isshown in low magnification (original ×4), Figures A, E, G, I, K and B,D, F, H, J and L are shown in higher magnifications (original ×20 and×40, respectively).

FIG. 6A is a summarizing bar graph of single marker staining in humanadult kidneys (HAK). Data were calculated as average % of expressingcell±SD. Each marker was tested in 3 HAK.

FIG. 6B is a representative dot plot graph of CD24 and CD133 co-stainingdemonstrating a large fraction of CD24⁺CD133⁺ cells in HAK. Quadrateswere placed according to the isotype control confiding the negativestaining to the lower left quadrant. Percentage of cells for each markercombination appears in the quadrant.

FIG. 7 is a hypothetical model of regional identity of human fetalkidney cells according to changes in surface marker expression duringdifferentiation of the nephric-lineage. Note that CD24 or CD133 can bealso added as a third marker to NCAM⁺EpCAM⁻ NCAM⁺EpCAM⁺ populations andas such represent putative stem/progenitor cell populations.Abbreviations: MM, metanephric mesenchyme; CM, condensed mesenchyme; LM,loose mesenchyme; UB, ureteric bud.

FIGS. 8A-Q show expression of selected genes in NCAM+EpCAM−, NCAM+EpCAM+as compared to NCAM− populations.

FIGS. 9A-Q are graphs illustrating gene expression analysis in sortedNCAM/EpCAM subpopulations. Quantitative reverse transcription-polymerasechain reaction (qRT-PCR) analysis of (A-E) renal stem/progenitor genes(Six2, Cited1, Sall1, Wt1 and Pax2), (F-G) vimentin and E-cadherin (H-L)‘sternness’ genes (β-catenin/CTNNB1, EZH2, BMI1, Nanog and Oct4) and(M-Q) surface marker (FZD7, ACR2B, NTRK2, CD24 and CD133) geneexpression in NCAM/EpCAM magnetically separated cells from HFK (15-19weeks of gestation). Normalization was performed against control HPRTexpression and RQ calculated relative to the NCAM− fraction. Data werecalculated as average±SD of at least 3 independent samples. ***P<0.001,*P<0.05 versus NCAM−. Sall1 expression in NCAM⁺ EpCAM⁺ cells was nearsignificance (p<0.059).

FIGS. 10A-G are graphs illustrating gene expression analysis in sortedPSA−NCAM subpopulations. Quantitative reverse transcription-polymerasechain reaction (qRT-PCR) analysis of (FIGS. 10A-E) renal stem/progenitorgenes (Six2, Citedl, Sall1, Wt1 and Pax2), (FIGS. 10 F-G) vimentin andE-cadherin genes expression in PSA−NCAM magnetically separated cellsfrom HFK (15-19 weeks of gestation). Normalization was performed againstcontrol HPRT expression and RQ calculated relative to the PSA−NCAM⁻fraction. Data were calculated as average±SD of at least 3 independentsamples. ***P<0.001, *P<0.05 versus PSA−NCAM⁻. Sall1 expression in NCAM⁺EpCAM⁺ cells was near significance (p<0.059).

FIGS. 10H-N are graphs illustrating gene expression analysis in NCAMsubpopulations as measured by quantitative reversetranscription-polymerase chain reaction (qRT-PCR).

FIGS. 11A-B are graphs illustrating assessment of clonogenic capacitiesof isolated HFK cells sorted according to NCAM and PSA−NCAM surfacemarkers. Data of sorted (11A) NCAM and (11B) PSA−NCAM⁺ cells show theirhigh clonogenic potential in all concentrations.

FIGS. 12A-B are graphs and photographs illustrating the results of thelimiting dilution assay which was performed on HFK cells sortedaccording to NCAM+EpCAM−, NCAM+EpCAM+ and NCAM−EpCAM+ and NCAM−EpCAM−.All cell fractions were plated in 96-well micro well plates at 0.3, 1, 3and 5 cells per well dilution. The number of colonized wells wasrecorded after 3-4 weeks. NCAM+EpCAM− cells show highest clonogenicpotential and to a lesser extent NCAM+EpCAM+ fraction compared toNCAM−EpCAM+ and NCAM−EpCAM− cells which formed no clones.

FIG. 13A is a graph illustrating that ALDH+ HFK cells have increasedclonogenic capabilities.

FIGS. 13B-E are graphs illustrating elevated expression levels of renalprogenitor genes in ALDH+ sorted cells compared to ALDH− HFK cells.

FIGS. 14A-B are bar graphs comparing the effect of serum free medium andserum containing medium on surface marker expression levels in humanfetal kidney cells.

FIGS. 15A-E are bar graphs comparing the effect of serum free medium andserum containing medium on nephric progenitor gene expression levels(FIGS. 15A-C), E-cadherin levels (FIG. 15D) and FoxD1 levels whichrepresents stromal differentiation (FIG. 15E) in human fetal kidneycells.

DESCRIPTION OF EMBODIMENTS OF THE INVENTION

The present invention, in some embodiments thereof, relates to isolatedpopulations of renal stem cells and methods of isolating and using same.

Before explaining at least one embodiment of the invention in detail, itis to be understood that the invention is not necessarily limited in itsapplication to the details set forth in the following description orexemplified by the Examples. The invention is capable of otherembodiments or of being practiced or carried out in various ways.

Nephrogenesis takes place in a discrete anatomic compartment termed themetanephric mesenchyme (MM) which is comprised of self-renewing renalstem cells that give rise to all cell types of the nephron as well as topediatric renal cancer (Wilms' tumor) and may prove valuable for renalregeneration after their isolation.

Renal failure, whether arising from an acute or chronic decline in renalfunction, is a severe condition that can result in substantial orcomplete failure of the filtration, reabsorption, endocrine andhomeostatic functions of the kidney. It is therefore desirable to obtaincells such as stem cells capable of developing into cells that couldsupply some or all of the functions of the kidney.

While reducing the present invention to practice, the present inventoridentified cell surface progenitor markers in human fetal kidney (HFK)which provides for a signature for the isolation of renalstem/progenitor cells. Such a characterization is a major step in theuse of stem cells in clinical settings.

Thus, as is illustrated herein below and in the Examples section whichfollows, the present inventor have used FACS and immunostaining toperform comprehensive profiling of surface antigens up-regulated in amicroarray study in both the developing kidney and blastema-enrichedstem-like Wilm's tumor xenografts.

No marker was specifically localized to the MM. Nevertheless, FZD7 andNTRK2 were preferentially localized to the nephrogenic zone (MM andemerging tubules), comprised <10% of HFK cells and were mostly presentwithin the EpCAM⁻ and EpCAM^(dim) fractions, indicating putativestem/progenitor markers. In contrast, single markers such CD24 and CD133as well as double-positive CD24⁺CD133⁺ cells comprise >50% of HFK cellsand predominantly co-express EpCAM^(bright), indicating they are mostlymarkers of differentiation. Furthermore, identification of NCAM1(interchangeably used with NCAM) exclusively in the MM and in MM-derivednephron progenitor structures but also in stroma assisted the presentinventors in pinpointing the presence of subpopulations that areputative MM-derived progenitor cells (NCAM⁺EpCAM⁺FZD7⁺), MM stem cells(NCAM⁺EpCAMTZD7⁺) or both (NCAM⁺FZD7⁺).

These results provide a feasible approach for experimental cell sortingof human renal progenitors as well as a framework for developing cellselection strategies for renal cell-based therapies.

In addition, the present inventor showed that NCAM⁺EpCAM⁻ cells highlyoverexpressed most MM stem genes (FIGS. 9A-E). Expression of MM stemgenes were reduced in sorted NCAM⁺EpCAM⁺ (containing putative MM-derivedprogenitor cells) compared to NCAM⁺EpCAM⁻ cells but still higher incomparison with the NCAM⁻ cell fraction, indicating a hierarchy forenrichment for the renal ‘progenitor’ genes. Furthermore, enhancedclonogenic capacity was found for sorted NCAM+ and PSA−NCAM+ cells(FIGS. 11A-B), indicating the presence of stem cells.

Whilst further reducing the present invention to practice, the presentinventor unexpectedly found that culturing of fetal renal cells in aserum free medium (SFM) allows for the enrichment of progenitor cells(FIGS. 15A-F). Prior sorting of the fetal renal cells to NCAM⁺subpopulations (FIGS. 12A-B) or ALDH+/ALDH^(bright) subpopulations(FIGS. 13A-F) enhanced the clonogenic potential of the cells and stemcell specific marker expression thereof.

Thus according to an aspect of the present invention there is providedan isolated population of cells comprising at least 50%, 60%, 70%, 80%,90% or more say 100% renal stem cells having a EpCAM−/FZD7+ signature.Such cells are cells composing the metanephric mesenchyme (MM, see FIG.7) of the renal cortex.

As used herein, the term “isolated” means that a cell population isremoved from its natural environment. As used herein, the term“purified,” means that a cell population is essentially free from anyother cell type (e.g., feeder fibroblasts).

As used herein the term “stem cells” refers to cells which maydifferentiate to all cell types of the nephron and are typically locatedin the MM.

As used herein “progenitor cells” can differentiate to certain type ofcells in the nephron and are typically located outside the MM.

According to an exemplary embodiment the renal stem cells have aEpCAM−/FZD7+/NCAM− signature. Such cells may be of the loose mesenchyme(LM) in the renal cortex.

According to an exemplary embodiment the renal stem cells have aEpCAM−/FZD7+/NCAM+ signature (or NCAM+/EpCAM−/CD133+/CD24+). Such cellsmay be of the condensed mesenchyme (CM) in the renal cortex.

NCAM+ populations of the present invention further comprise a geneexpression profile as provided in FIGS. 8C-Q. Assaying expression of anyof the genes of the provided expression profile may be used to qualifycells of the NCAM+, NCAM+EpCAM signature.

According to a further aspect of the present invention there is providedan isolated population of cells comprising at least 50%, 60%, 70%, 80%,90% or more say 100% MM-derived progenitor cells having a NCAM+/EpCAM+signature.

According to an exemplary embodiment the MM-derived progenitor cellsfurther express a surface marker selected from the group consisting ofFZD7, NTRK2 and PSA−NCAM1 as well as ROR2, ACVR2B, CD133 and CD24).These cells typically compose the C and S shape bodies of the fetalkidneys and may differentiate to the nephric tissue (e.g., tubules andglumeruli-visceral and parietal epithelium). These cells are abundant inthe fetal kidney but in the tissues of the collective system.

According to a further aspect of the present invention there is providedan isolated population of cells comprising at least 50%, 60%, 70%, 80%,90% or more say 100% fetal renal cells having aNCAM−/EpCAM+/CD24+/CD133+ signature. These cells are differentiatednephrons.

According to a further aspect of the present invention there is providedan isolated population of cells comprising at least 50%, 60%, 70%, 80%,90% or more say 100% renal stromal cells having a NCAM+/FZD7− signature.These cells can differentiate to the interstitium (whereby cells of theinterstitium comprise NCAM+/EpCAM− signature).

According to an exemplary embodiment the renal stromal cells have aEpCAM−/NCAM+/FZD7− signature.

According to a further aspect of the present invention there is providedan isolated population of cells comprising at least 50%, 60%, 70%, 80%,90% or more say 100% uretric bud cells having a EpCAM+/FZD7+ signature.

According to an exemplary embodiment the ureteric bud cells have aEpCAM+/FZD7+/NCAM− signature. These cells may differentiate todifferentiated cells of the collecting ducts.

Thus, according to a further aspect of the present invention there isprovided an isolated population of cells comprising at least 50%, 60%,70%, 80%, 90% or more say 100% fetal renal cells having aNCAM−/EpCAM+/FZD7− signature.

According to an exemplary embodiment the cells are derived from a fetus,e.g., human fetus. Typically, the nephrogenic zone exists 5-34 weeks ofhuman gestation and cells can be isolated along that time frame.According to an exemplary embodiment the cells are retrieved from ahuman fetal kidney of mid gestation 14-21 weeks.

As used herein the phrase “renal stem cell” refers to a cell which isnot terminally differentiated as a renal cell but which has the abilityto differentiate into specialized cell having one or more structuraland/or functional aspects of a physiologic kidney. According to specificembodiments the renal stem cells are not embryonic stem cells.

The present invention further provides for a method of isolating theaforementioned cells. This is effected by enriching for a subpopulationof renal cells from a renal tissue (e.g., fetal), the subpopulation ofrenal cells having any of the above-mentioned surface-marker signature.

Thus a human kidney (e.g., fetal) is provided. The kidney may comprise awhole kidney or fragments thereof (e.g., renal capsule).

Below is a list of some of the exemplary markers of the presentinvention with their accession numbers.

NCAM1 (3 variants): NM_(—)181351, NM_(—)000615, NM_(—)001076682; EPCAM:NM_(—)002354; FZD7: NM_(—)003507; CD24: NM_(—)013230; CD133 (PROM1):NM_(—)006017; NTRK2: AF410902; PSA−NCAM, Polysialylated NCAM1 same ID asNCAM1; ACVRIIB: NM_(—)001106; ROR2 (2 variants): M97639 NM,_(—)004560;oct4 (POU5F1): NM_(—)203289 NM_(—)002701; six2: NM_(—)016932 {accessionnumber: AF136939; sall1: NM_(—)002968; ctnnb1 NM_(—)001098210(NM_(—)001098209 XM_(—)001133660 XM_(—)001133664 XM_(—)001133673XM_(—)001133675 NP_(—)001091679 XP_(—)001133660 XP_(—)001133664XP_(—)001133673 XP_(—)001133675); vimentin: NM_(—)003380 (accessionnumber: M14144); Bmil: NM_(—)005180 (accession number BC011652); ezh2 (2variants): NM_(—)152998 NM_(—)004456; nanog: NM_(—)024865 (accessionnumber: AB093576 (complete); aqp1—NM_(—)000385 (accession number:M77829); aqp3: NM_(—)004925; e-cadherin (CDH1): NM_(—)004360 (accessionnumber: L08599).

Antibodies for the above mentioned cell markers are commerciallyavailable. Examples include but are not limited to, NCAM1 (eBioscience),EPCAM (MiltenyiBiotec), FZD7 (R&D Systems), CD24 (eBioscience), CD133(MiltenyiBiotec), NTRK2 (R&D Systems), PSA−NCAM (MiltenyiBiotec) ACVRIIB(R&D Systems), ROR2 (R&D Systems).

As used herein, the term “enriching” refers to a procedure which allowsthe specific subpopulation of renal cells to comprise at least about50%, preferably at least about 70%, more preferably at least about 80%,about 95%, about 97%, about 99% or more renal stem cells having thedesired signature (e.g. EpCAM−/FZD7+ or NCAM+/EpCAM+).

The enriching may be effected using known cell sorting procedures suchas by using a fluorescence-activated cell sorter (FACS).

As used herein, the term “flow cytometry” refers to an assay in whichthe proportion of a material (e.g. renal cells comprising a particularmaker) in a sample is determined by labeling the material (e.g., bybinding a labeled antibody to the material), causing a fluid streamcontaining the material to pass through a beam of light, separating thelight emitted from the sample into constituent wavelengths by a seriesof filters and minors, and detecting the light.

A multitude of flow cytometers are commercially available including fore.g. Becton Dickinson FACScan and FACScalibur (BD Biosciences, MountainView, Calif.). Antibodies that may be used for FACS analysis are taughtin Schlossman S, Boumell L, et al, [Leucocyte Typing V. New York: OxfordUniversity Press; 1995] and are widely commercially available.

It will be appreciated that the enriching may also be effected bydepleting of non-relevant subpopulations such as renal stromal cells orinterstitium (interstitial) cells having a cell surface signature asdescribed herein.

Once isolated, cells of the present invention may be cultured andallowed to proliferate in serum free medium (SFM) in order to preservetheir stem/progenitor cell phenotype. Optionally the cells may bedirected to differentiate into a desired lineage.

The present inventors have found that culturing fetal renal cells in SFMallows for the enrichment of a renal progenitor cell population, asevidenced by enhancement of stem-cell associated genes and enhancementof clonogenicity (see Examples 3 and 4 herein below). The presentinventors showed that serum containing media results in unwanted effectsof stromal expansion at the expense of stem/progenitor cells.

Thus, according to another aspect of the present invention there isprovided a method of enriching for renal stem cells, the methodcomprising culturing a population of fetal renal cells in a culturemedium devoid of serum, thereby enriching for renal stem cells.

A contemplated culture medium is IMDM (Invitrogen) or DMEM (Invitrogen).

According to one embodiment, the fetal renal cells are culturedfollowing a limiting dilution assay, where a single cell is culturedindividually in a single container (e.g. a single cell is cultured inone well of a 96 well plate).

Pre-selecting for a particular cell population prior to culture inserum-free medium may aid in enhancing the purity of the isolated stemcell populations. Thus the present invention contemplates pre-selectingfetal renal cells which have a NCAM+signature, a NCAM+/EpCAM− signature,a NCAM+/EpCAM+ signature or an ALDH+ signature.

According to another embodiment a particular cell population may beselected following culture in SFM. Thus the present inventioncontemplates post-selecting fetal renal cells which have a NCAM+signature, a NCAM+/EpCAM− signature, a NCAM+/EpCAM+ signature or anALDH+ signature.

Accordingly, the present invention contemplates pure populations (morethan 80%, more than 85%, more than 90%, more than 91%, more than 92%,more than 93%, more than 94%, more than 95%, more than 96%, more than97%, more than 98%, more than 99%, more than 99.5%, or even 100%) ofrenal stem and/or progenitor cells having a NCAM+signature, aNCAM+/EpCAM+ signature, NCAM+/EpCAM− or an ALDH+ signature.

In order to confirm the presence of renal stem cells, the cells may betested for expression of stem cell-specific genes. An upregulation ofsuch genes infers the presence of renal stem cells. Such genes include,but are not limited to Six2 (NM_(—)016932-accession number: AF136939),osr1 (NM_(—)145260.2), Pax2 (NM_(—)003987.3 NM_(—)000278.3,NM_(—)003988.3, NM_(—)003989.3, NM_(—)003990.3), Sall1 (NM_(—)002968)and Cited 1 (NM_(—)001144885.1, NM_(—)001144886.1, NM_(—)001144887.1NM_(—)004143.3). Methods for analyzing for the expression of stemcell-specific genes include RT-PCR, Northern blot, Western blot, flowcytometry and the like. Since clonogenicity is a function of stem cells,another way to confirm the presence of renal stem cells is to analyzethe clonogenic potential of the cells, as described in Example 3, hereinbelow.

Cells of the present invention can be genetically modified to express atransgene. This may be used to increase survival of the cells, renderthem immortalized or differentiated to a desired lineage. Examples ofsuch transgenes and methods of introducing the same are provided below.

Candidate genes for gene therapy include, for example, genes encodingthe alpha 5 chain of type IV collagen (COL4A5), polycystin,alpha-galactosidase A, thiazide-sensitive sodium chloride cotransporter(NCCT), nephrin, actinin, or aquaporin 2.

Further, genes encoding erythropoietin or insulin can be introduced intoa kidney stem cell. For treatment of anemia associated with renalfailure or diabetes it can be useful to introduce into a patient a stemcells modified to express erythropoietin or insulin. The renal stemcells can be stably or transiently transfected with DNA encoding anytherapeutically useful polypeptide.

The renal stem cells of the invention can also be provided with atransgene encoding VEGF or some other factor that can promote growth andor differentiation of cells.

These genes can be driven by an inducible promoter so that levels ofenzyme can be regulated. These inducible promoter systems may include amutated ligand binding domain of the human estrogen receptor (ER)attached to the protein to be produced. This would require that theindividual ingest tamoxifen to allow expression of the protein.Alternatives are tetracyclin on or off systems, RU486, and a rapamycininducible system. An additional method to obtain relatively selectiveexpression is to use tissue specific promoters. For instance, one couldintroduce a transgene driven by the KSP-cadherin, nephrin oruromodulin-specific promoter.

Cells isolated by the method described herein can be geneticallymodified by introducing DNA or RNA into the cell by a variety of methodsknown to those of skill in the art. These methods are generally groupedinto four major categories: (1) viral transfer, including the use of DNAor RNA viral vectors, such as retroviruses (including lentiviruses),Simian virus 40 (SV40), adenovirus, Sindbis virus, and bovinepapillomavirus for example; (2) chemical transfer, including calciumphosphate transfection and DEAE dextran transfection methods; (3)membrane fusion transfer, using DNA-loaded membrane vesicles such asliposomes, red blood cell ghosts, and protoplasts, for example; and (4)physical transfer techniques, such as microinjection, electroporation,or direct “naked” DNA transfer. Cells can be genetically altered byinsertion of pre-selected isolated DNA, by substitution of a segment ofthe cellular genome with pre-selected isolated DNA, or by deletion of orinactivation of at least a portion of the cellular genome of the cell.Deletion or inactivation of at least a portion of the cellular genomecan be accomplished by a variety of means, including but not limited togenetic recombination, by antisense technology (which can include theuse of peptide nucleic acids, or PNAs), or by ribozyme technology, forexample. Insertion of one or more pre-selected DNA sequences can beaccomplished by homologous recombination or by viral integration intothe host cell genome. The desired gene sequence can also be incorporatedinto the cell, particularly into its nucleus, using a plasmid expressionvector and a nuclear localization sequence. Methods for directingpolynucleotides to the nucleus have been described in the art. Thegenetic material can be introduced using promoters that will allow forthe gene of interest to be positively or negatively induced usingcertain chemicals/drugs, to be eliminated following administration of agiven drug/chemical, or can be tagged to allow induction by chemicals(including but not limited to the tamoxifen responsive mutated estrogenreceptor) for expression in specific cell compartments (including butnot limited to the cell membrane).

Calcium phosphate transfection, which relies on precipitates of plasmidDNA/calcium ions, can be used to introduce plasmid DNA containing atarget gene or polynucleotide into isolated or cultured cells. Briefly,plasmid DNA is mixed into a solution of calcium chloride, then added toa solution which has been phosphate-buffered. Once a precipitate hasformed, the solution is added directly to cultured cells. Treatment withDMSO or glycerol can be used to improve transfection efficiency, andlevels of stable transfectants can be improved usingbis-hydroxyethylamino ethanesulfonate (BES). Calcium phosphatetransfection systems are commercially available (e.g., ProFection fromPromega Corp., Madison, Wis.). DEAE-dextran transfection, which is alsoknown to those of skill in the art, may be preferred over calciumphosphate transfection where transient transfection is desired, as it isoften more efficient.

Since the cells of the present invention are isolated cells,microinjection can be particularly effective for transferring geneticmaterial into the cells.

The developmental potential of stem cells thus obtained can beinvestigated using methods which are well known in the art. For exampleby injection into other organs (liver, muscle, heart and bone marrow) totest their multipotency Clarke et al. describes protocols forinvestigating the development potential of stem cells (Clarke et al.2000 Science 288:1660).

The renal stem cells of the invention can be used to supplement orsubstitute for kidney cells that have been destroyed or have reducedfunction. Thus, they can be used to treat patients having poor or nokidney function. The renal stem cells of the invention or cells derivedfrom the renal stem cells of the invention may be capable of performingthe filtration and reabsorptive/secretive functions of the kidney.

Thus according to an aspect of the present invention there is provided amethod of treating a renal damage in a subject in need thereofcomprising administering to the damaged kidney of the subject atherapeutically effective amount of any of the isolated population ofcells, thereby treating the renal disease in the subject.

Cells of the present invention can be used to treat any form of acute orchronic kidney disease, diabetic nephropathy, renal disease associatedwith hypertension, hypertensive acute tubular injury (ischemic, toxic),interstitial nephritis, congenital anomalies(Aplasia/dysplasia/obstructive uropathy/reflux nephropathy); hereditaryconditions (Juvenile nephronophtisis, ARPCKD, Alport, Cystinosis,Primary Hyperoxaluria); Glomerulonephritides (Focal SegmentalGlomerulosclerosis); Multisystem Diseases (SLE, HSP, HUS).

The cells may be administered per se or as part of a pharmaceuticalcomposition where they are mixed with a suitable carrier or excipient.

As used herein a “pharmaceutical composition” refers to a preparation ofone or more of the active ingredients described herein with otherchemical components such as physiologically suitable carriers andexcipients. The purpose of a pharmaceutical composition is to facilitateadministration of a compound to an organism.

Herein the term “active ingredient” refers to the renal progenitor cells(or cells differentiated therefrom) accountable for the biologicaleffect.

Hereinafter, the phrases “physiologically acceptable carrier” and“pharmaceutically acceptable carrier” which may be interchangeably usedrefer to a carrier or a diluent that does not cause significantirritation to an organism and does not abrogate the biological activityand properties of the administered compound. An adjuvant is includedunder these phrases.

Herein the term “excipient” refers to an inert substance added to apharmaceutical composition to further facilitate administration of anactive ingredient. Examples, without limitation, of excipients includecalcium carbonate, calcium phosphate, various sugars and types ofstarch, cellulose derivatives, gelatin, vegetable oils and polyethyleneglycols.

The renal stem cells or cells derived from the renal stem cells can beadministered into a subject such as surgically or by infusion. Forexample, renal stem cells are injected in vivo into a kidney that is inthe postischemic recovery phase. This can be tested easily in an animalmodel predictive of ischemic kidney damage, the renal pedicle of ananesthetized mouse is clamped for 30 minutes to induce kidney ischemia.Renal stem cells are then injected into the juxtamedullary region(approximately 2000 cells at a depth of 2-4 mm). After 2 weeks ofrecovery, immunohistochemical analysis is used as described above tolook for differentiated cells surface markers GP330, Tamm-Horfall,Dolichos Biflorous, and the like. Post-incorporation differentiationstatus can then be compared to pre-injection marker status.

The stem cells of the invention, or cells derived from the stem cells ofthe invention (e.g., epithelial cells endothelial cells, mesangialcells, vascular smooth muscle cells, and pericytes) can be used toconstruct artificial kidney systems. Such a system can be based on ahollow fiber filtration system.

In one example of a filtration device, the stem cells of the inventionor differentiated progeny thereof are grown on the interior of hollowfibers having relatively high hydraulic conductivity (i.e.,ultrafiltration coefficient). The hollow fiber passes through a chamberthat is provided with a filtrate outlet port. Arterial blood containingmetabolic wasterand other unwanted material is introduced into one endof the hollow fiber through an inlet port. Blood passed through thefiber and exits the other end of the fiber through an outlet port whereit passed into the patient's vascular venous flow. As blood passesthrough the fiber, filtrate pass through the cells lining the interiorof the fiber and through the hollow fiber itself. This filtrate thenpasses out of the chamber containing the fiber through the filtrateoutlet port. The device preferably includes many such hollow fibers eachof which can be in its own chamber. Alternatively many, many hollowfibers (100-100,000 or even more) can be bundled together in a singlechamber.

The cells of the invention can be used to create a tubule-processingdevice. In such a device the stem cells of the invention ordifferentiated cells derived from the stem cells of the invention can begrown in a layer on the exterior of the semipermeable hollow fiber. Thefiber is placed in a chamber that is provided with an inlet port and anoutlet port. As ultrafiltrate from filtered blood flows through thechamber, reabsorbant passes through the cell layer and through the wallof the fiber into the lumen of the fiber from which it can be directedback into the patient's systemic circulation. Ultrafiltrate that is notreabsorbed passes through the outlet port of the chamber.

In the devices described above, it can be desirable to coat the fibersurface that will bear the cell layer with extracellular matrixcomponents. For example, the fiber can be coated with materials such ascollagen (e.g., Type I collagen or Type IV collagen), proteoglycan,fibronectin, and laminin or combinations thereof. It can be desirable tocombine various cell types on the inner or outer surface of the fibers.For example, it can be desirable to include endothelial cells andpericyte, vascular smooth muscle cells or mesangial cells or fibroblastsor combinations thereof. It can also be useful to provide a feeder layerof cells, e.g., irradiated fibroblasts or other cells that can providesoluble factors and structural support to cells they are indirectly ordirectly in contact with.

The above-described filtration system and the above-described tubuleprocessing system can be combined to create an artificial kidney. Suchsystems are described in U.S. Pat. No. 6,150,164, hereby incorporated byreference. A number of suitable materials for forming the hollow fiberare described in U.S. Pat. No. 6,150,164, hereby incorporated byreference.

The present invention provides a method of using renal stem cells orprogenitor cells to characterize cellular responses to biologic orpharmacologic agents involving isolating the cells as described s,culture expanding the cells to establish a plurality of MRPC cultures,contacting the MRPC cultures with one or more biologic or pharmacologicagents, identifying one or more cellular responses to the one or morebiologic or pharmacologic agents, and comparing the one or more cellularresponses of the cultures. Tissue culture techniques known to those ofskill in the art allow mass culture of hundreds of thousands of cellsamples from different individuals, providing an opportunity to performrapid screening of compounds suspected to be, for example, teratogenicor mutagenic.

As used herein the term “about” refers to ±10%.

The terms “comprises”, “comprising”, “includes”, “including”, “having”and their conjugates mean “including but not limited to”. This termencompasses the terms “consisting of” and “consisting essentially of”.

The phrase “consisting essentially of” means that the composition ormethod may include additional ingredients and/or steps, but only if theadditional ingredients and/or steps do not materially alter the basicand novel characteristics of the claimed composition or method.

As used herein, the singular form “a”, “an” and “the” include pluralreferences unless the context clearly dictates otherwise. For example,the term “a compound” or “at least one compound” may include a pluralityof compounds, including mixtures thereof.

Throughout this application, various embodiments of this invention maybe presented in a range format. It should be understood that thedescription in range format is merely for convenience and brevity andshould not be construed as an inflexible limitation on the scope of theinvention. Accordingly, the description of a range should be consideredto have specifically disclosed all the possible subranges as well asindividual numerical values within that range. For example, descriptionof a range such as from 1 to 6 should be considered to have specificallydisclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numberswithin that range, for example, 1, 2, 3, 4, 5, and 6. This appliesregardless of the breadth of the range.

Whenever a numerical range is indicated herein, it is meant to includeany cited numeral (fractional or integral) within the indicated range.The phrases “ranging/ranges between” a first indicate number and asecond indicate number and “ranging/ranges from” a first indicate number“to” a second indicate number are used herein interchangeably and aremeant to include the first and second indicated numbers and all thefractional and integral numerals therebetween.

As used herein the term “method” refers to manners, means, techniquesand procedures for accomplishing a given task including, but not limitedto, those manners, means, techniques and procedures either known to, orreadily developed from known manners, means, techniques and proceduresby practitioners of the chemical, pharmacological, biological,biochemical and medical arts.

As used herein, the term “treating” includes abrogating, substantiallyinhibiting, slowing or reversing the progression of a condition,substantially ameliorating clinical or aesthetical symptoms of acondition or substantially preventing the appearance of clinical oraesthetical symptoms of a condition.

It is appreciated that certain features of the invention, which are, forclarity, described in the context of separate embodiments, may also beprovided in combination in a single embodiment. Conversely, variousfeatures of the invention, which are, for brevity, described in thecontext of a single embodiment, may also be provided separately or inany suitable subcombination or as suitable in any other describedembodiment of the invention. Certain features described in the contextof various embodiments are not to be considered essential features ofthose embodiments, unless the embodiment is inoperative without thoseelements.

Various embodiments and aspects of the present invention as delineatedhereinabove and as claimed in the claims section below find experimentalsupport in the following examples.

EXAMPLES

Reference is now made to the following examples, which together with theabove descriptions, illustrate some embodiments of the invention in anon limiting fashion.

Generally, the nomenclature used herein and the laboratory proceduresutilized in the present invention include molecular, biochemical,microbiological and recombinant DNA techniques. Such techniques arethoroughly explained in the literature. See, for example, “MolecularCloning: A laboratory Manual” Sambrook et al., (1989); “CurrentProtocols in Molecular Biology” Volumes I-III Ausubel, R. M., ed.(1994); Ausubel et al., “Current Protocols in Molecular Biology”, JohnWiley and Sons, Baltimore, Md. (1989); Perbal, “A Practical Guide toMolecular Cloning”, John Wiley & Sons, New York (1988); Watson et al.,“Recombinant DNA”, Scientific American Books, New York; Birren et al.(eds) “Genome Analysis: A Laboratory Manual Series”, Vols. 1-4, ColdSpring Harbor Laboratory Press, New York (1998); methodologies as setforth in U.S. Pat. Nos. 4,666,828; 4,683,202; 4,801,531; 5,192,659 and5,272,057; “Cell Biology: A Laboratory Handbook”, Volumes I-III Cellis,J. E., ed. (1994); “Culture of Animal Cells—A Manual of Basic Technique”by Freshney, Wiley-Liss, N.Y. (1994), Third Edition; “Current Protocolsin Immunology” Volumes I-III Coligan J. E., ed. (1994); Stites et al.(eds), “Basic and Clinical Immunology” (8th Edition), Appleton & Lange,Norwalk, Conn. (1994); Mishell and Shiigi (eds), “Selected Methods inCellular Immunology”, W. H. Freeman and Co., New York (1980); availableimmunoassays are extensively described in the patent and scientificliterature, see, for example, U.S. Pat. Nos. 3,791,932; 3,839,153;3,850,752; 3,850,578; 3,853,987; 3,867,517; 3,879,262; 3,901,654;3,935,074; 3,984,533; 3,996,345; 4,034,074; 4,098,876; 4,879,219;5,011,771 and 5,281,521; “Oligonucleotide Synthesis” Gait, M. J., ed.(1984); “Nucleic Acid Hybridization” Hames, B. D., and Higgins S. J.,eds. (1985); “Transcription and Translation” Hames, B. D., and HigginsS. J., eds. (1984); “Animal Cell Culture” Freshney, R. I., ed. (1986);“Immobilized Cells and Enzymes” IRL Press, (1986); “A Practical Guide toMolecular Cloning” Perbal, B., (1984) and “Methods in Enzymology” Vol.1-317, Academic Press; “PCR Protocols: A Guide To Methods AndApplications”, Academic Press, San Diego, Calif. (1990); Marshak et al.,“Strategies for Protein Purification and Characterization—A LaboratoryCourse Manual” CSHL Press (1996); all of which are incorporated byreference as if fully set forth herein. Other general references areprovided throughout this document. The procedures therein are believedto be well known in the art and are provided for the convenience of thereader. All the information contained therein is incorporated herein byreference.

Example 1 Determination of Cell Surface Progenitor Markers in HumanFetal Kidneys

Materials and Methods

Establishment of a primary culture from human fetal kidney (HFK): HFKsamples were retrieved from aborted fetuses.

Collected human fetal kidney cells (HFK) were washed with cold HBSS(Invitrogen, Carlsbad, Calif., USA) and minced into ˜1 mm cubes usingsterile surgical scalpels. The dissected tissue was then incubated for 2hours at 37° C. with Iscoves's Mod Dulbecco's Medium (IMDM) (Invitrogen)supplemented with 0.1% collagenase II (Invitrogen). The digested tissuewas then gradually forced through a 100 μm, 70 μm and 50 μm cellstrainer to achieve a single cell suspension, and after removal of thedigesting medium resuspended in growth medium [IMDM containing 10% fetalbovine serum (Invitrogen), 100 ng/ml EGF, 100 ng/ml bFGF and 10 ng/mlSCF (R&D Systems, Inc, Minneapolis, USA)] and plated in flasks. Cellswere incubated at 37° C. and 5% CO₂. Medium was replaced every day forthe first 2 days and then every 3-4 days. Cells were passed uponreaching confluence using 0.05% Trypsin/EDTA (Invitrogen) to detach themfrom the plate. Cells were passed for up to 3 passages andcryo-preserved in FBS with 10% DMSO (Sigma-Aldrich, St Louis, Mo., USA).

IHC staining of HFK. Immunostaining was performed as previouslydescribed²¹. Briefly, 4 μm sections of HFKs (12 or 19 weeks ofgestation) were mounted on super frost/plus glass (Menzel, Glazer,Braunschweig, Germany) and processed by the labeled—(strept)avidin-biotin (LAB-SA) method using a histostain plus kit (Zymed, SanFrancisco, Calif., USA). Heat-induced antigen retrieval was performed bycontrolled microwave treatment using an H2800 model processor (EnergyBean Sciences, INC) in 10 mM citrate buffer, PH 6.0 for 10 min at 97° C.The sections were treated with 3% H₂O₂ for 10 minutes and stained forEZH2 (Zymed), CD56 (Ancell Corporation), CD90 (AbD serotec), DLK1 (RayBiotec), CD24, GPR39, CD133 (abcam), SIX2 (ABNOVA), FZD7, FZD2 (NOVUSbiologicals), ACRIIB and NTRK1 (R&D Systems). Negative controlincubations were performed by substituting non-immune serum for theprimary antibody. Biotinylated second antibody was applied for 10minutes followed by incubation with horseradish peroxidase—conjugatedstreptavidin (HRP-SA) for 10 minutes. Following each incubation, theslides were washed thoroughly with Optimax wash buffer (Biogenex). Theimmunoreaction was visualized by an HRP-based chromogen/substratesystem, including DAB (brown) chromogen (liquid DAB substratekit—Zymed). The sections were then counterstained with Mayer'shematoxylin, dehydrated and mounted for microscopic examination.Antibody details are provided in Table 1, herein below.

TABLE 1 Marker Antibody Identified Manufacturer Catalog # Rabbit antiEZH2 EZH2 Zymed, SKU#36-6300 San Francisco, CA Monoclonal anti-humanCD56 Ancell Corporation, 208-020 CD56 (NCAM1) Bayport, MN, USA Mouseanti-human CD90 CD90, AbD serotec, MCA90 thy 1 Kidlington, Oxford, UKMouse anti- Preadipocyte DLK1, Ray Biotec, Inc, NR-08-0034 factor-1PREF1 Parkway Lane, Norcross GA Mouse monoclonal CD24 CD24 abcam,ab31622 Cambridge, UK. Six2 monoclonal antibody SIX2 ABNOVA, H000010736-Walnut, USA M01 Rabbit polyclonal anti- FZD7 NOVUS biologicals, NLS4900frizzled-7 Littleton, USA Rabbit polyclonal anti- FZD2 NOVUSbiologicals, NLS3488 frizzled-2 Littleton, USA Monoclonal anti-humanACRIIB R&D Systems, Inc, MAB3393 Activin RIIB antibody Minneapolis, USARabbit polyclonal to GPR39 abcam, ab39283 GPCR GPR39 Cambridge, UK.Rabbit polyclonal to CD133 abcam, ab16518 CD133 Cambridge, UK.Monoclonal anti-human NTRK1 R&D Systems, Inc, MAB3971 TrkB antibodyMinneapolis, USA

Flow cytometry. Cells were detached from culture plated withnon-enzymatic cell dissociation solution (Sigma-Aldrich) and a viablecell number was determined using Trypan blue assay (Invitrogen). Cells(1×10⁵ in each reaction) were suspended in 50 μl of FACS buffer [0.5%BSA and 0.02% sodium azide in PBS (Sigma-Aldrich and Invitrogen,respectively)] and blocked with FcR Blocking Reagent (MiltenyiBiotec)and human serum (1:1) for 15 minutes at 4° C. Cells were then incubatedfor 45 minutes with a primary antibody for CD24, NCAM1, C-KIT (all fromeBioscience), Thy-1, CD90 (both from BD Pharmingen), CD34, CD133, EpCAM,PSA−NCAM (all from MiltenyiBiotec), ACVR2B, FZD7 or NTRK1 (all from R&DSystems) or a matching isotype control.

Antibodies used in the flow cytometry assays are provided in Table 2,herein below.

TABLE 2 Marker Isotype Antibody identified control Manufacturer Catalog# CD24-PE CD24 Mouse eBioscience San 12-0247 IgG1 Diego, USA Biotinanti- CD24 Mouse eBioscience 13-0247 human CD24 IgG1 FITC anti- CD34Mouse MiltenyiBiotec 130-081- human CD34 IgG2a 001 PE anti-human CD56NCAM1 Mouse eBioscience 12-0569 (N-CAM, NCAM1) IgG2a,κ FITC mouse Thy-1Mouse BD Biosciences, 555595 anti-human CD90 IgG1,κ San Jose, USACD133/1 CD133 Mouse MiltenyiBiotec 130-090- (AC133)-APC IgG1 826 CD326EpCAM Mouse MiltenyiBiotec 130-080- (EpCAM)- FITC IgG1 301 MonoclonalACR2B Mouse R&D Systems, MAB3393 anti-human Activin IgG1 Inc. RIIBantibody Biotinylated anti- FZD7 rat IgG2A R&D Systems, BAM1981human/mouse Inc. Frizzled-7 antibody FITC mouse anti- CD90 Mouse BDPharmingen 555595 Human CD90 IgG1,κ Affinity Purified C-KIT MouseeBioscience 141179 antihuman IgG1,κ CD117 (cKit) Monoclonal anti- NTRK1Mouse R&D Systems, MAB3971 human TrkB IgG1 Inc. antibody Anti-PSA- PSA-Mouse MiltenyiBiotec 130-093- NCAM-PE NCAM IgM 274

Cells were washed with FACS buffer, and incubated for 30 minutes at 4°C. with a secondary Ab if needed [Avidin-Fluorescein, APC Streptavidin(both from BD Biosciences) or Alexa Fluor 647 goat anti mouse AlexaFluor 488 goat anti mouse (both from Invitrogen)]. Cells viability wastested using 7AAD viability staining solution (eBio science).

Details of secondary Abs or S/A conjugated enzymes used in flowcytometry assays are provided in Table 3 herein below.

TABLE 3 Reagent Manufacture company # Avidin-Fluorescein (Avidin-FITC)R&D Systems, Inc. F0030 APC Streptavidin BD Biosciences. 554067 AlexaFluor 647 goat anti mouse Invitrogen A31625 Alexa Fluor 488 goat antimouse Invitrogen A31620

Cell's labeling was detected using FACSCalibur (BD). Flow cytometryresults were analyzed using FlowJo analysis software. Viable cells weredefined by their FSC/SSC profiles and, in addition, their lack of 7AAD.Analysis of EpCAM subpopulations was performed by gating cell fractionsaccording to EpCAM staining intensity (negative, dim or bright) versusFSC. The second marker was then examined in each subpopulation gate.When triple staining was performed, the EpCAM subpopulation wasinitially gated and then co-staining of the other two markers in eachsubpopulation was examined.

Results

SIX2: Of the multiple regulatory genes specifying renal progenitors,SIX2 is a transcription factor that has been shown in mice to specifyself-renewing epithelial renal stem cells that have the ability to giverise to all cell types in the nephron⁶. Immunostaining of mid-gestationhuman fetal kidney (FK) revealed localization of such SIX2-expres singcells to the metanephric mesenchyme (MM), specifically to the capmesenchyme (CM), where renal stem cells are suggested to reside^(6, 8)(FIGS. 1A, 1B). While unsuitable for human cell sorting, SIX2 staininghighlights the location of the desired putative MM stem cells.

EpCAM (CD326). The Epithelial Cell Adhesion Molecule (EpCAM) isexpressed virtually on all normal epithelia in vertebrates²² and cantherefore serve as a marker for epithelial differentiation. Accordingly,Trzpis et al²³ have recently shown that in mid-gestation human FK (by 10weeks of gestation), hEpCAM was expressed by the ureteric bud (UB) andcomma-shaped (C) and S-shaped (S) bodies, whereas the MM did not expresshEpCAM. Moreover, they found differential hEpCAM staining levels duringnephrogenesis, where the weakest staining for hEpCAM was observed in thecomma- and S-shaped bodies, which are progenitor nephron derivatives ofthe MM and higher levels in the UB and developing tubules of thenephron, indicating a correlation between hEpCAM levels and the degreeof epithelial differentiation. The present inventors examined cellpopulations of low-passage human FK cells by flow cytometry and revealedthat 80.0±11.2% of the cells express EpCAM (FIG. 2A). This resultcorrelated with its wide-spread distribution in epithelial cells of thedeveloping kidney. Moreover two subpopulations within the EpCAMpopulation, EpCAM^(dim) and EpCAM^(bright) were detected, suggestive ofepithelial progenitor and more differentiated tubular cells,respectively (FIGS. 3A-Y). A clearer separation between EpCAM^(dim) andEpCAM^(bright) cell populations was noted in older HFK.

NCAM1 (CD56). NCAM1 transcript levels were up-regulated in both human FKand stem-like WT xenografts (>three-fold increment)¹⁶. Immunostaining ofsections of mid-gestation human FK (14-20 week) demonstrated predominantstaining in the nephrogenic zone and renal stroma, while mature tubuleswere devoid of staining. In the nephrogenic zone, we observed strongexpression in the CM, similar to SIX2 and also in early S and commashaped nephron figures (i.e., MM and its derivatives) and newly formingtubules but not in UBs (FIGS. 1A-J). This staining pattern of NCAM1 hasbeen observed in the developing mouse kidney^(24, 25). Examination ofpopulations of low-passage human FK cells by single staining flowcytometry revealed that 29.1±8.2% of the cells express NCAM1 (FIG. 2B),representing nephrogenic zone and stroma-derived NCAM expressing cells.Two sub-populations of NCAM cells, NCAM⁺EpCAM⁻ (13.5±4.9% of totalcells) and NCAM⁺EpCAM⁺ (14.5±3.7% of total cells) were also detected.Because EpCAM is not expressed in the stroma or in the MM, theNCAM⁺EpCAM⁻ subpopulation is indicative of cells originating from bothof these areas, while NCAM⁺EpCAM⁺ cells are a heterogeneous pool ofprogenitor cells from the nephrogenic zone, including newly developedtubules. This sub-population could be further separated intoNCAM⁺EpCAM^(dim) and NCAM⁺EpCAM^(bright) cell fractions ((FIGS. 3B-E)).In the EpCAM^(dim) population a significantly larger fraction consistsof NCAM expressing cells compared to that found in the EpCAM^(bright)cell fraction (P<0.0001) (FIGS. 3B-E), further indicating NCAM as anepithelial progenitor marker. Taking into account that in thenephrogenic zone low levels of EpCAM were previously noted in theimmediate MM-derived structures (S- and comma-shaped) and higher levelsin emerging tubules, the NCAM⁺EpCAM^(dim) cells possibly represent theformer. In addition, the present inventors have analyzed the long chainform of polysialic acid (PSA) characteristic of the low adhesiveembryonic form of NCAM (PSA−NCAM), the probe of which was not includedin the microarrays¹⁶. This surface marker closely resembles NCAM'sstaining pattern (various developmental stages including condensed MM,renal vesicles, the distal portion of S-shaped bodies, and primitivetubules) but is not detected in the renal stroma. Accordingly, PSA−NCAMwas found to be expressed in 8.6±3.2% of HFK cells (FIG. 2E) and to peakin the EpCAM^(dim) cell fraction (P<0.015 compared to the EpCAM^(bright)cell fraction) (FIGS. 3F-I). Furthermore, PSA⁺EpCAM⁻ and PSA⁺EpCAM⁺ cellfractions are more limited in expression by comparison to NCAM/EpCAM(2.3±1.3% and 4.2±0.9% of total cells, respectively). Interestingly,when applying triple staining for PSA, NCAM and EpCAM the putative MMcell fraction, NCAM⁺PSA⁺EpCAM⁻ was found to be expressed in 2.5±2.2% oftotal cells, while NCAM⁺PSA⁺EpCAM⁺ from later developmental stages in4.3±0.3% of total cells (FIGS. 4C-D), indicating that PSA and NCAMlocalize in similar progenitor areas. NCAMTSA⁺EpCAM⁻ cells could not bedetected.

Frizzled 2,7 (FZD2, FZD7). Both transcript levels of FZD2 and FZD7 (Wntreceptors) were up-regulated in both human FK and stem-like WTxenografts¹⁶. Recently, activation of the Wnt/β-catenin pathway has beenshown to maintain the progenitor pool in the metanephric mesenchyme²⁷.Thus, FZDs represent surface marker molecules that may have a functionalrole in maintaining progenitor cells. Immunostaining of sections ofmid-gestation human FK (14-20 week) revealed that while FZD2demonstrated widespread expression (FIGS. 5A-L), staining all of thetubular cells, FZD7 was detected predominantly in the nephrogenic zone,staining all cell types in that area [MM (both loose and condensedmesenchyme), UBs, early nephron figures, newly forming tubules] but notat all in renal stroma (FIGS. 1E-F). Correlating with its reservedlocalization, FZD7 was detected in only 9.5±3.7% of the HFK cells (FIG.2F). Examination of the FZD7 expressing cells in relation with EpCAMsub-populations, showed that largest fractions of FZD7⁺ cells existswithin the EpCAM^(neg) and EpCAM^(dim) fraction and to a much lesserextent in the EpCAM^(bright) cell fraction (P<0.02) (FIGS. 3J-L). Thus,while EpCAM⁺FZD7⁻ cells represent the largest fraction (53.8±13.4% oftotal cells), FZD7⁺EpCAM⁺ cells were observed (3.9±1.2% of total cells)which likely represent MM- and UB-derived progenitors and FZD7⁺EpCAM⁻cells (2.5±0.6% of total cells), which may originate solely from the MM.Furthermore, using triple FACS staining of HFK cells that also includesNCAM (FIGS. 4G-H) the present inventors were able to demonstrate cellpopulations of the FZD7⁺EpCAM⁺NCAM⁺ progenitor phenotype (MM-derived,2.5±1.0% of total cells, 4.7±1.0% FZD7⁺NCAM⁺ cells within the EpCAMpopulation) as well as FZD7⁺NCAM⁺EpCAM⁻ (0.6±0.5% of total cells,2.2±0.7% FZD7⁺NCAM⁺ cells within the EpCAM⁻ population) and surprisinglyalso FZD7⁺EpCAM⁻NCAM⁻ phenotypes (2.0±0.8% of total cells, 7.8±4.18%FZD7⁺NCAM⁻ cells within the EpCAM⁻ population), which are both likely torepresent putative MM-originating stem cells.

Activin receptor JIB (ACVRIIB). ACVRIIB qualified as a microarraypredicted marker. Interestingly, mice lacking ACVRIIB show abnormalitiesin kidney development and in anterior/posterior patterning of the axialskeleton show abnormalities^(28, 29), further emphasizing functionalimportance in the renal progenitor population. Similar to NCAM and FZD7,in the sections of human FK, ACVRIIB was preferentially localized to thenephrogenic zone, showing strong expression in all structure types(blastema, UBs, comma- and S-shaped bodies and also developing tubules).ACVRIIB was also detected in parietal epithelium of fetal glomeruli butnot on stromal cells (FIGS. 1G-H). A similar staining pattern wasobserved by in-situ hybridization of E14.5 mouse kidneys (robustexpression of ActRIIB mRNA in the condensed metanephric mesenchyme,differentiating nephrons and UB branches). While according to itslocalization ACVRIIB has potential as a renal progenitor marker, FACSanalysis of HFK cells showed extremely varying expression levels andprecluded its further investigation.

NTRK2. NTRK2 qualified as a microarray predicted marker as similar toFZD7 it was up-regulated in microarrays of WT-stem like tumors and humanFK. Previous analysis of the developing mouse kidney showed NTRK2 tolocalize to the MM while in WT NTRK2 has been suggested as a badprognostic marker³⁰. Immunostaining of the human FK showed NTRK2 tolocalize to cells within the MM but also to early differentiation stagesin the nephrogenic zone and some differentiated tubules but not stroma(FIGS. 1I-J). FACS analysis revealed NTRK2 to stain 12.1±3.4% of thehuman FK (FIG. 2C). Analysis of NTRK2 according to EpCAM subpopulationsrevealed a tendency towards higher expression levels in both thenegative and dim fraction compared to the bright one (FIGS. 3N-Q). Tofurther strengthen the presence of progenitor phenotypes the presentinventors found by triple staining of NTRK2 along with NCAM and EpCAM,EpCAM⁺NCAM⁺NTRK2⁺ cells (3.1±2.5% of total, 6.8±3.3% NCAM⁺NTRK2⁺ cellswithin the EpCAM population) as well as putative MM stem cellpopulations, EpCAM⁻NCAM⁺NTRK2⁺ cells (0.61±0.3% of total, 3.3±2.5%NCAM⁺NTRK2⁺ cells within the EpCAM⁻ population) and EpCAM_NCAM⁻NTRK2⁺cells (2.7±2.4% of total, 7.5±2.7% NCAM⁻NTRK2⁺ cells within the EpCAM⁻population) (FIGS. 4K-L).

GPR39, DLK1. These markers, up-regulated in microarrays of both human FKand stem-like WT xenografts, were found to be ubiquitously expressed indifferentiated renal tubular epithelial cells in sections of human FKwhile only faintly positive or negative in progenitor structures of thenephrogenic zone and were therefore eliminated from FACS analysis (FIG.5C-D).

CD34. CD34 is a well known marker of hematopoietic stem cells (HSC)³¹.FACS analysis demonstrated CD34 to be expressed in 14.4±12.9% of HFKcells. Immunostaining for the CD34 protein specifically demonstratedwidespread endothelial localization (glomerular and peri-tubular) in allparts of the human FK (FIG. 5G-H), including in the nephrogenic zonewhereas CM and other epithelial progenitor structures are devoid of CD34expression. CD34 is therefore not an epithelial stem cell marker in thehuman FK but rather a marker for vascular differentiation. c-Kit, anadditional hematopoietic stem cell marker, was not detected in the humanFK cells.

CD90. Antigenic phenotypes of adult MSC consistently include CD90 andCD105³². In addition, CD90 was shown to be broadly expressed onheterogeneous rat fetal kidney cells transplanted to injured kidneys¹³.Immunolocalization of CD90 in the human FK revealed predominantexpression in renal tubular cells but not in the nephrogenic zone (FIG.5I-J) and 25.3±8.5% of HFK cells expressed CD90.

CD24. CD24 was not differentially expressed in the developing humankidneys or in WT stem-like xenografts. Nevertheless, the previousdemonstration of CD24 as characteristic of the molecular phenotype ofrenal progenitor cells in the developing mouse kidneys³³, as well as theutilization of CD24 (along with CD133) to specify human renal progenitorcells³⁴ from developing human kidneys, led the present invnetors toexamine its expression. Immunostaining of human FK showed widespreadexpression and localized CD24 to mature tubules (renal stroma was devoidof CD24) (FIGS. 5K-L). Accordingly, FACS analysis demonstrated thatapproximately 73.6±20.6% of HFK cells express CD24 (FIG. 2H). Whenanalyzed in regard with EpCAM sub-populations, the abundance of CD24expressing cells increases along epithelial differentiation (in contrastwith for instance FZD7) so that approximately 80% of the EpCAM^(bright)cells are CD24⁺ cells (P<0.0001 compared to CD24⁺ cells found in the dimand negative fractions) (FIG. 3R-U), indicating that CD24 ispredominantly a marker of differentiation in the human FK. Moreover,triple staining with NCAM revealed that CD24 is expressed in low levelsonly in putative MM fractions; CD24⁺NCAM⁺EpCAM⁻ and CD24⁺NCAM⁻EpCAM⁻cell fractions (2.0±1.2%, 3.7±2.8% of total cells, respectively) incontrast to a CD24⁺NCAM⁻EpCAM⁺ differentiated phenotype (34.1±14.6% oftotal cells) (FIGS. 4E-F). Thus, sorting cells from the human FKaccording to CD24 would result in a heterogeneous population comprisedpredominantly of differentiated cells and to a much lesser extent ofstem/progenitor cells.

CD133. Although the biological function of CD133 remains unknown, CD133is recognized as a stem cell marker for normal and cancerous tissues³⁵.Indeed, CD133 alone or in a combination with other markers is currentlyused for the isolation of stem cells from numerous tissues, such as bonemarrow, brain, prostate, liver, pancreas³⁵⁻³⁸, and both developing andadult kidney (along with CD24)³⁴′³⁹. Among adult organs, the kidney hasbeen reported to have large numbers of CD 133⁺cells^(35, 40). Aspreviously shown for the fetal pancreas, detect CD133 positivity inhuman fetal kidney tissue could not be detected. However, FACS analysisof human FK cells demonstrated that 56.9±15.8% of the cells expressCD133 (FIG. 2I). Furthermore, the EpCAM^(bright) fraction contained thelargest population of CD133 expressing cells with significantly smallerpopulations in EpCAM^(dim) and EpCAM^(neg) cells (P<0.0001) (FIGS.3V-Y). In addition, similar to CD24, triple FACS staining demonstrated alarge population of CD133⁺EpCAM⁺NCAM⁻ cells (29.5±10.6% of total cells)and a relatively small ones of the CD133⁺NCAM⁺EpCAM⁺ (14.4±4.5% of totalcells) and CD133⁺NCAM⁺EpCAM⁻ putative progenitor and stem phenotypes(1.1±1.2% of total cells) (FIGS. 4I-J). Because CD24⁺CD133⁺ cells havebeen recently suggested a renal ‘stem cell’ fraction³⁴, the presentinventors analyzed expression of CD133 in conjunction with CD24. Doublestaining showed that the CD24⁺CD133⁺ fraction comprises 55.5±6.4% of thehuman FK cells, while triple staining with EpCAM showed that within theEpCAM^(bright) fraction approximately 60% of the cells are CD24⁺CD133⁺and to a much lesser extent in the EpCAM^(dim) and EpCAM^(neg) cellfractions (P<0.0001) (FIGS. 4I-J). Thus, similar to cells expressing theCD24 marker, most of the CD133⁺ cells in the human FK and alsoCD133⁺CD24⁺ cells are of a differentiated tubular phenotype and are notin any way exclusive to the stem/progenitor pool.

Marker expression in the human adult kidney. Renal cell progenitormarkers are expected to decrease once maturation occurs. The presentinventors therefore analyzed cell surface marker expression in the humanadult kidney (HAK). FACS analysis of HAK cells for single markerexpression revealed reduced PSA−NCAM, FZD7, NTRK2 and NCAM levelscompared to HFK, indicative of a progenitor origin (FIGS. 6A-B). Incontrast, similar and even increased expression levels in the HAK wereobserved for CD105, CD90, CD133 and CD24 (FIGS. 6A-B). Moreover,CD24⁺CD133⁺ cells represent a large cell fraction in the HAK, comprising64.26±10.15% of the total cells.

Discussion

In the present example, the present inventor has analyzed for theexpression of putative stem cell markers in the human fetal kidney.Using comprehensive immunocytochemical and flow cytometric analysis ofhuman FK cells, the expression profile of a variety of surface antigenswere characterized, some of which are considered markers oforgan-specific stem cells and the others have been recently suggested toappear on malignant renal stem/progenitor cells of wilms' tumors and inhuman FK¹⁶. Given the similarities in molecular marker expression inprogenitors from wilms' tumors and the developing human kidney, itappears likely that these cell populations are derivatives of the samelineage.

The present data suggest that none of these putative stem cell markersare restricted to kidney-specific epithelial stem/progenitor cells, buton the contrary, stem cell markers are always also expressed ondifferentiated elements. The necessity for marker combination is shownnot only by lack of specific staining of the nephrogenic mesenchyme butalso by high percentage of expression of single markers in human FKcells, over 50% of cells for markers such as CD24 and CD133, as well asthe relative high marker abundance within the EpCAM^(bright) fraction.Because CD24 and CD133 mostly qualify as markers for identification ofdifferentiated tubular cells, their combination will not enrich for aprogenitor phenotype. More relevant for the enrichment ofstem/progenitor cells is the utilization in combination of at least oneof the markers that were found to localize predominantly to thenephrogenic zone and to a much lesser extent to differentiated epithelia(NCAM, PSA−NCAM, FZD7, and NTRK2). Interestingly, using a highlyreliable antibody the present inventors have recently identified NCAM asa candidate marker for the renal malignant progenitor population ofwilms' tumor⁴¹. Because NCAM is not at all expressed on UBs ordifferentiated epithelia it can be extremely useful for positiveselection of MM-derived progenitor nephron populations (NCAM⁺X⁺) if thesecond marker is clearly not detected on MM and stromal cells. Thisdefinition is most suitable for the NCAM⁺EpCAM⁺ fraction which wasdetected among the human FK cells. Moreover, because EpCAM isdifferentially expressed in the nephrogenic zone²³, identification ofthe NCAM⁺EpCAM^(dim) subset, possibly pinpoints an earlier MM-derivedprogenitor population (FIG. 7). Second markers that are expressed in allparts of the nephrogenic zone and are not detected on stromal cellspotentially produce populations that include both MM-stem cells and aheterogeneous MM-derived progenitor population of the nephrogenic zone.This includes a wide variety of second marker combination, such as therather small and discrete populations of NCAM⁺FZD7⁺ or NCAM⁺NTRK2⁺ cellswhich were identified, but potentially also larger NCAM⁺CD24⁺ andNCAM⁺CD133⁺ cell populations (if indeed CD133 will be directly shown notto localize to stromal cells).

The rarities of putative MM-stem cells arising from condensates isdemonstrated by triple FACS staining of these cell populations withEpCAM and analysis for those populations that totally lack epithelialdifferentiation (EpCAM^(neg)). In all cases these were the smallestpopulations by comparison to EpCAM expressing fraction, showingNCAM⁺FZD7⁺EpCAM⁻, NCAM⁺NTRK2⁺EpCAM⁻ and NCAM⁺CD133⁺EpCAM⁻ cell fractionsto be ≦1% of HFK cells, and NCAM⁺PSA⁺EpCAM⁻ ˜2.5% of the cells.Interestingly, within the EpCAM^(neg) fraction there were NCAMTZD7⁺ orNCAM⁻NTRK2⁺ but not NCAMTSA⁺ cells. These findings correlate withstaining patterns in which FZD7 and NTRK2 also localize to loosemesenchyme (LM) while PSA appears with condensation, possibly indicatingthe former fractions to arise from LM (FIG. 7).

In practice, cell sorting according to two positive markers and onenegative is likely to be cumbersome and therefore eliminating EpCAMafter positively selecting for a single marker that is expressedexclusively along the developmental stages of renal epithelia (MM,MM/UB-derived progenitors, developing and developed tubules but notstroma) might be more practical for sorting MM-enriched stem cells. Inthis setting, using an initial marker that localizes preferentially tothe nephrogenic zone as opposed to a predominantly marker ofdifferentiation is advantageous. One such potential combination includesthe very small but consistent population of FZD7⁺EpCAM⁻ or PSA⁺EpCAM⁻cells. In any event, the relative paucity of stem/progenitor phenotypeshighlights the need for early sorting of human FK cells according tomarker molecules followed by their expansion in vitro rater thanapplication of multipassage culture of unsorted heterogeneous human FKcells for cell selection⁴².

The profiling of renal surface antigens initiated here forms the basisfor exploring other markers and for investigating the function ofsuggested progenitor cell sub-populations in the renal context (FIG. 7).

Example 2 HFK Cell Sub Populations Sorted According to Specific MarkersRetain in Culture Molecular Aspects of Regional Identity and StemnessProfile

Because immunostaining of HFK demonstrated that the markers areregionally specified, the present inventor wanted to verify thatregional differences are maintained in HFK cells. As aproof-of-principle sorted NCAM⁺EpCAM⁻, NCAM⁺EpCAM⁺ (containing putativeMM stem- and MM-derived progenitor cells, respectively) were comparedwith NCAM⁻ HFK cell populations as NCAM and EpCAM are important surfacemarkers for the present characterization system.

Materials and Methods

Magnetic cell sorting: At least three independent kidney samples wereused for sorting of NCAM/EpCAM as well as PSA−NCAM subpopulations.Sorted cells were of primary cultures established from the same HFK usedin the FACS analysis of progenitor marker expression. Cells weredetached with Trypsin/EDTA and resuspended in growth medium. Cells weretransferred trough 30 μm Pre-Separation Filter (Miltenyi Biotec GmbH,Bergisch Gladbach, Germany) then washed and resuspended in pH 7.2 MACSbuffer (0.5% BSA, 2 mM EDTA in PBSX1). Cells were magnetically labeledwith NCAM1 (CD56) MultiSort MicroBeads kit (Miltenyi Biotec GmbH)according to the manufacturer's instructions and positive labeled cells(NCAM⁺) were enriched with LS Columns. CD56 MicroBeads were releasedfrom the cells with MultiSort Release Reagent (Miltenyi Biotec GmbH) andCD56 positive cells were further separated with EpCAM (CD326) positiveand negative cells using CD326 MicroBeads (Miltenyi Biotec GmbH) on LSColumns according to the manufacturer's instructions. Enrichment ofcells to CD56 and CD326 was validated using flow cytometry.

Quantitative reverse transcription-PCR: Sorted NCAM⁺EpCAM⁻,NCAM⁺EpCAM⁺and NCAM⁻ sub-populations of HFK were tested for theexpression of:

1. Transcription factors specifying renal stem/progenitor cells in theMM (SIX2, CITED1, SALL1, WT1, PAX2) (Cho E A, Dressler G R. San Diego:San Diego: Academic Press; 2003. In The Kidney: From Normal Developmentto Congenital Disease. pp. 195-210; Cicero S A, et al. Proc Natl AcadSci USA. 2009; 106(16):6685-6690);

2. The marker pair Vimentin/E-cadherin that are expressed in earlystages of kidney development during mesenchymal (Vim+) to epithelial(E-cad+) conversion and differentiation (Cho E A, Dressler G R. SanDiego: San Diego: Academic Press; 2003. In The Kidney From NormalDevelopment to Congenital Disease. pp. 195-210; Cicero S A, et al. ProcNatl Acad Sci USA. 2009; 106(16):6685-6690).

3. ‘Stemness’ genes (Wnt pathway, β-catenin; Polycomb group, EZH2, BMI1)4. Pluripotency genes (NANOG, OCT4) and

5. Surface markers (ACR2B, FZD7, NTRK2, CD133 and CD24). In addition,sorted PSA−NCAM⁺ and PSA−NCAM⁻ HFK cells were analyzed for theexpression of genes included in groups 1 and 2. Total RNA from cells wasisolated using RNeasy Micro Kit (Qiagen GmbH, Hilden, Germany) accordingto the manufacturer's instructions. cDNA was synthesized using HighCapacity cDNA Reverse Transcription kit (Applied Biosystems, CaliforniaUSA) on total RNA. Real-time PCR was performed using an ABI7900HTsequence detection system (Perkin-Elmer/Applied Biosystems) in thepresence of TaqMan Gene Expression Master Mix (Applied Biosystems). PCRamplification was performed using gene specific TaqMan Gene ExpressionAssay-Pre-Made kits (Applied Biosystems). PCR results were analyzedusing SDS RQ Manager 1.2 software. Statistical analysis was performedusing a non-paired 2-tails T-test. Statistical significance wasconsidered at P<0.05.

Results

Although a heterogeneous cell population, NCAM⁺EpCAM⁻ cells highlyoverexpressed (>five fold) most MM stem/progenitor genes in fiveseparate HFK (FIGS. 9A-E), levels of which were already reduced in theNCAM⁺EpCAM⁺ cell fraction (presumably more differentiated), but stillhigher (Wt1, Sall1) in comparison with the NCAM⁻ cell fraction,indicating a hierarchy for enrichment for the renal ‘progenitor’ genes.Considerably lower E-cad levels were observed for the NCAM⁺EpCAM⁻ andNCAM⁺EpCAM⁺ cell fractions, while NCAM⁺EpCAM⁻ also significantlyoverexpressed vimentin (FIGS. 9F-G). In addition, while there was atendency for elevation of the ‘stemness genes’ in the NCAM⁺ fractions,only β-catenin achieved significance in NCAM⁺EpCAM⁻ cells (FIGS. 9H-L),most likely due to large variations across human samples. Finally,analysis of surface marker expression in the sorted sub-populationsshowed elevated FZD7, ACVRIIB and NTRK2 in the NCAM⁺ fractions (bothFZD7 and NTRK2 genes significantly overexpressed in the NCAM⁺EpCAM⁻fractions) as opposed to CD24 and especially CD133 (FIGS. 9M-Q). Similarresults were found when analyzing expression in sorted PSA−NCAM⁺ cellsby comparison to the negative fraction. PSA−NCAM (see before) showedsignificant enrichment for Six2, Sall1, Wt1 and Pax2 (FIGS. 10A-E and 10H-I, J and M)) as well as reduced levels of E-cadherin (FIGS. 10E-G and10L), all indicative of a stem/progenitor origin. Thus, HFK cells retainaspects of regional identity as determined by marker immunostaining.

Example 3 HFK Cell Sub Populations Sorted According to Specific MarkersShow Enhanced ‘Stemness’ Function

Following verification of the renal ‘stemness’ gene profile in humanrenal stem/progenitors sorted according to specific markers, the presentinventors analyzed these fractions for clonogenic ability, an importantfeature of stem/progenitor cells.

Materials and Methods

Limiting dilution assay was performed on HFK cells sorted according toNCAM and PSA−NCAM. Both positive and negative fractions were plated in96-well micro well plates at 0.3, 1, 3 and 5 cells per well dilution.The number of colonized wells was recorded after 3-4 weeks.

In addition HFK cells were sorted according to ALDH expression and theclonogenic ability was tested in serum containing medium and serum freemedium.

Flow cytometry: Flow cytometry was performed as described in Example 1,herein above. Detection of cells with high ALDH1 enzymatic activity wasperformed using the ALDEFLUOR kit (StemCell Technologies, Durham, N.C.,USA).

Results

Enhanced clonogenic capacity was found for sorted NCAM+ and PSA−NCAM+cells (FIGS. 11A-B). In addition it was found that when culturing sortedcells in serum free media, a media that preserves epithelial kidneystem/progenitor cells the entire clonogenic capabilities of HFK cellsare within the NCAM+fraction, both NCAM+EpCAM− and NCAM+EpCAM+ (thefirst has an advantage over the second) stem/progenitor fractions butnot in differentiated NCAM− fraction (both EpCAM+ or EpCAM−) (FIGS.12A-B).

Enhanced clonogenic capacity was found for sorted ALDH+/bright cells(FIG. 13A) compared to ALDH− cells.

Furthermore ALDH+/bright sorted cells showed enhanced expression ofrenal progenitor genes compared with ALDH^(neg) cells as measured by qRTPCR (FIGS. 13B-E).

Example 4 HFK Cell Sub Populations Cultured in Serum Free MediumPreserves Expression of Stem-Cell Associated Markers

Materials and Methods

Culturing of HFK cells: Cells were grown in DMEM:F12, a 1:1 mixture ofHam's F12 and high-glucose Dulbecco's modified Eagle medium supplementedwith 1% non essential Amino acids, 1% of sodium pyruvate (all fromInvitrogen, Carlsbad, Calif., USA), 1% N2 supplement 100×, 0.4% B27supplement (both from Gibco, Carlsbad, Calif., USA), 0.2% Lipid mixture,1% growth factor mixture containing 2% glucose 30%, 200 mg transferring,50 mg insulin, 0.1% sodium selanite 0.3 mM, 0.01% progesterone 2 Mm and19.33 mg putrescine (all from Sigma-Aldrich, St Louis, Mo., USA), 4μg/ml heparin, supplemented with 10 ng/ml FGF, 20 ng/ml EGF (R&DSystems, Inc, Minneapolis, USA). For passage, cells cultured inserum-free media were dissociated with Cell dissociation solution(Sigma-Aldrich) without trypsin.

Experiments were Preformed on Low Passages

qRT-PCR analysis: RT-PCR analysis was performed as described in Example2, herein above.

Results

HFK cells cultured in serum free medium showed different expressionlevels surface markers compared to HFK cells cultured in serumcontaining medium as illustrated in FIG. 14A. FIG. 14B illustrates thathigh expression of particular markers (namely CD24, CD133 and EPCAM) ispreserved following 5 passages in serum free medium; whereas expressionof NCAM is increased following 5 passages.

In addition, HFK cells cultured in serum free medium showed elevatedexpression levels of nephric progenitor genes (FIGS. 15A-C), compared toHFK cells. E-cadherein expression was more rapidly lost in serumcontaining medium than serum free medium (FIG. 15D) and FoxD1(indicative of stromal cells) expression was shown to be elevated inserum containing medium compared to serum free medium (FIG. 15E).

Although the invention has been described in conjunction with specificembodiments thereof, it is evident that many alternatives, modificationsand variations will be apparent to those skilled in the art.Accordingly, it is intended to embrace all such alternatives,modifications and variations that fall within the spirit and broad scopeof the appended claims.

All publications, patents and patent applications mentioned in thisspecification are herein incorporated in their entirety by referenceinto the specification, to the same extent as if each individualpublication, patent or patent application was specifically andindividually indicated to be incorporated herein by reference. Inaddition, citation or identification of any reference in thisapplication shall not be construed as an admission that such referenceis available as prior art to the present invention. To the extent thatsection headings are used, they should not be construed as necessarilylimiting.

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Methods Cell Biol 86, 241-255 (2008).

1. A method of isolating renal stem cells, the method comprisingenriching for a subpopulation of renal cells from a fetal renal tissue,said subpopulation of renal cells having a signature selected from thegroup consisting of an NCAM+ signature, an EpCAM−/FZD7+ signature, anEpCAM+/FZD7+ signature, an NCAM−/EpCAM+/FZD7− signature and anNCAM−/EpCAM+/CD24+/CD133+ signature wherein said enriching is effectedsuch that at least 80% cells are of said subpopulation of renal cells.2-3. (canceled)
 4. The method of claim 1, wherein when said signature isan NCAM+ signature, the method further comprises isolating cells havingan EpCAM−/NCAM+ signature.
 5. The method of claim 1, wherein when saidsignature is an NCAM+ signature, the method further comprises isolatingcells having an EpCAM+/NCAM+ signature.
 6. The method of claim 1,further comprising culturing said subpopulation of renal cells in serumfree medium following said enriching. 7-9. (canceled)
 10. The method ofclaim 1, wherein when said signature is an EpCAM−/FZD7+ signature themethod further comprises isolating cells having an EpCAM−/FZD7+/NCAM−signature.
 11. The method of claim 1, wherein when said signature is anEpCAM−/FZD7+ signature the method further comprises isolating cellshaving an EpCAM−/FZD7+/NCAM+ signature 12-16. (canceled)
 17. The methodof claim 1, wherein when said signature is an NCAM+ signature, themethod further comprises isolating cells having a NCAM+/FZD7− signature.18-28. (canceled)
 29. An isolated population of fetal cells comprising apopulation selected from the group consisting of a population whichcomprises at least 80% fetal renal stem cells having a EpCAM−/FZD7+signature, a population which comprises at least 80% fetal renal stemcells having a NCAM+ signature, a population which comprises at least80% MM-derived fetal progenitor cells having a NCAM+/EpCAM+ signature, apopulation which comprises at least 80% fetal renal stromal cells havinga NCAM+/FZD7− signature and a population which comprises at least 80%fetal ureteric bud cells having a EpCAM+/FZD7+ signature.
 30. Theisolated population of cells of claim 29, wherein when said populationcomprises at least 80% fetal renal stem cells having a EpCAM−/FZD7+signature, said renal stem cells have a EpCAM−/FZD7+/NCAM− signature.31. The isolated population of cells of claim 29, wherein when saidpopulation comprises at least 80% fetal renal stem cells having aEpCAM−/FZD7+ signature, said renal stem cells have a EpCAM−/FZD7+/NCAM+signature.
 32. (canceled)
 33. The isolated population of cells of claim29, wherein when said population comprises at least 80% fetal renal stemcells having a NCAM+ signature, said cells have a NCAM+ EpCAM−signature. 34-43. (canceled)
 44. A cell culture comprising a culturemedium and any of the isolated population of cells of claim
 29. 45. Thecell culture of claim 44, wherein said cells are seeded on a scaffold.46. A method of treating a renal damage in a subject in need thereofcomprising administering to the damaged kidney of the subject atherapeutically effective amount of any of the isolated population ofcells of claim 29, thereby treating the renal disease in the subject.47. A method of identifying an agent capable of regulatingdifferentiation of a renal stem cell, the method comprising contactingany of the isolated population of cells of claim 29 with an agent,wherein a change in developmental phenotype is indicative of the agentcapable of regulating differentiation of said renal stem cells. 48-53.(canceled)